EXPRESSION OF AN ENDO-IH,4-MANNANASE FROM BACILLUS SUBTILIS Gl IN E.COLI AND ITS PROPERTIES
Abstract
The bacterial strain Bacillus subtilis G1 producing an enzyme endo-β-l ,4-mananase was selected to create a recombinant mannanase. The gene encoding ndo-β-l ,4-mananase from B. subtilis G 1 was amplified with the specific primer pair MAF and MAR designed on the basis of the nucleotide sequence encoding the ndo-β-l ,4-mananase AF324506. The PCR product was cloned into the T/A cloning vector pTZS7R1T. The nucleotide sequence of the gene encoding ndo-β-l ,4-mananase with a length of 1101 bp was deposited in the GenBank: DQ309335. This gene was digested by two restriction enzymes Ncol and EcoRl and inserted into the expression vector pET22b( +) also cut by the same enzymes. The obtained recombinant plasmid pEMAG was transformed into E. coli BL21 resulted in the expression system E. coli BL2l/pEMAG. The recombinant ndo-β-l ,4-mananase had an optimal reaction temperature and pH at 45oC and pH 7.0, respectively. Organic solvents at the concentration of 10 - 30% all inhibited the enzyme, activity remained 35 - 82%.